1. Technical Field
The invention relates to the characterization of site-specific antibodies for O-GlcNAcylated proteins, and for the use of the characterized antibodies for the detection of O-GlcNAcylated proteins in biological systems, the screening of drugs that modulate protein O-GlcNAcylation, and the diagnosis and treatment of cancer.
2. Background Art
O-GlcNAcylation (N-acetylglucosamine modification) of nuclear and cytosol proteins regulates many cell functions. O-GlcNAcylation of proteins plays a critical role in cell cycle regulation, apoptosis and signal transduction. Modification of O-GlcNAcylation of proteins by O-GlcNAc transferase (OTG) (synthesis of O-GlcNAcylated protein) is reversible by the activity of O-GlcNAc hydrolase (O-GlcNAcase) (cleavage of an O-GlcNAc moiety of protein). The addition and removal of the O-GlcNAc moiety to serine (Ser) or threonine (Thr) regulate the function of proteins directly or via decreased phosphorylation of the same or nearby Ser and Thr residues, and are related to diabetes and cancer (1).
O-GlcNAcylated proteins play a critical role in physiology and in diseases including cancer. High O-GlcNAcase activity results in low O-GlcNAcylation of a critical site of a protein. Low O-GlcNAcylation of a critical site of an oncoprotein or tumor suppressor protein can increase phosphorylation at or near that site, and can thereby induce or promote cancer cell proliferation and/or metastasis.
Over 120 O-GlcNAcylated nuclear and cytosolic human proteins have been found in mammals. They include seven nuclear pore proteins, thirty-one transcription factors, six RNA-binding proteins, seven phosphatases, kinases and adapter proteins, twenty-nine cytoskeletal proteins, five chaperones, seven metabolic enzymes, other regulatory proteins and six viral proteins (2).
Contrary to the known consensus sequence of N-glycosylation, Asn-X-Ser/Thr (X=any amino acid except for Pro), the O-GlcNAcylation consensus sequence is not known. Most Ser/Thr sites for O-GlcNAcylation are on or near to phosphorylation sites. O-GlcNAcylation of a site prevents phosphorylation at the site or a site close to the O-GlcNAcylation site, reversing phosphorylation-dependent cellular function of the molecule. The converse mechanism also operates, with phosphorylation at or near a O-GlcNAcylation site preventing O-GlcNAcylation.
O-GlcNAcylation of a protein is carried out by activity of O-GlcNAc transferase using UDP-GlcNAc. O-GlcNAcylation can be reversed by activity of O-GlcNAcase. A single O-GlcNAcase gene, evolutionarily conserved from bacteria to has been found to carry out all O-GlcNAcase activity.
In contrast to the numerous phosphatases that regulate various critical cell functions including cell survival, proliferation and metastasis, only one O-GlcNAcase regulates the removal of an O-GlcNAc moiety from numerous proteins, and thereby regulate increased or decreased functions of cellular proteins. This fact strongly suggests that activity of the O-GlcNAcase depends on its substrate, which is an O-GlcNAcylation site (target amino acid sequence) unique to each protein. There is therefore a need for O-GlcNAcylation site-specific antibodies to detect the unique O-GlcNAcylation site characteristic of each protein.
Pan-specific antibodies for O-GlcNAcylation, which recognize only the O-GlcNAc moiety common to all O-GlcNAcylated protein cannot be used for identification of an O-GlcNAcylated target protein in Western blot analysis and immunohistochemistry (IHC). Even in immunoprecipitation (IP) assay, accuracy of the result depends on the capture antibodies. Even when the capture antibodies bind to the target proteins, the pan-specific antibodies for O-GlcNAcylation cannot distinguish the target O-GlcNAcylation site from other O-GlcNAcylation sites of the protein with differential cellular function.
There are multiple O-GlcNAcylated sites for each oncoprotein or tumor suppressor protein, whereas only one or two sites are involved with cancer cell proliferation and/or metastasis. It has been reported that the O-GlcNAcylation site of c-myc is Thr-58 (T58) and the counteracting phosphorylation sites are T58, Ser-62 (S62) and Ser-71 (S71) (3). The O-GlcNAcylation site of p53 has been reported to be Ser-149 (S149) and the counteracting phosphorylation site as Thr-155 (T155) (4).
For example, T58 of c-myc protein, a mutational hot spot in lymphomas, is a site for both phosphorylation and O-GlcNAcylation, which induce ubiquitin-dependent degradation and increased stability, respectively. T58/S62 phosphorylation of c-myc protein also decreases T58 GlcNAcylation (5,6). Another example is p53 tumor suppressor protein. Phosporylation of the p53 at T155 induces ubiquitin-dependent degradation, which decreases p53 levels, whereas GlcNAcylation of p53 at S149 prevents p53 degradation and stabilizes p53 proteins (4). Accumulation of the p53 protein is beneficial. Its role in cell growth-arrest and apoptosis in response to DNA damage protects cells from uncontrolled proliferation, which occurs in cancer cells. The need for O-GlcNAcylation site-specific antibodies for c-myc and p53 is therefore especially acute.
Inhibitors of GlcNAcase activity have been screened using bacterial and human recombinant GlcNAcases by measurement of a decreased fluorescent signal of the fluorescent substrate of GlcNAcase, 4-methylumbelliferol (4-MU)-GlcNAc, (pseudo substrate) which is cleaved by the enzyme resulting in a loss of fluorescent signal (7). This method can only identify inhibitors of GlcNAcase that inhibit 4-MU-GlcNAc cleavage. As previously stated, the specificity of O-GlcNAcase depends on the target O-GlcNAcylation site of a protein. A compound found to inhibit the cleavage of 4-MU-GlcNAc is therefore not necessarily an inhibitor of the action of O-GlcNAcase upon specific proteins, such as, p53 and c-myc. Thus, O-GlcNAcase inhibitors have to be screened using an actual O-GlcNAcylated protein as a substrate. This type of screening requires detection by site-specific antibodies. This is another reason why there is a need for O-GlcNAcylation site-specific antibodies to detect the unique O-GlcNAcylation site characteristic of each protein.